Silk Peptide For Improving Neuroprotective And Neurofunctional Effects And A Method Of Its Prepartion

ABSTRACT

A silk peptide having neuroprotective and neurofunctional activities and its preparation method are discussed. One method relates to preparing silk protein preferably having neuroprotective activity with weight average molecular weight of 200-100,000 by hydrolysis of silk fibroin; also discussed are a composition for preventing or treating brain disease comprising silk peptide and pharmaceutically acceptable carrier, and a composition for improving brain function.

TECHNICAL FIELD

The present invention relates to silk peptide having the effects ofneuroprotection and improving brain functions, and a method of itspreparation. More particularly, it relates to a method of preparing silkprotein preferably having neuroprotective activity with weight averagemolecular weight of 200-100,000 by hydrolysis of silk fibroin; acomposition for preventing or treating brain disease comprising silkpeptide and a pharmaceutically acceptable carrier; and a composition forimproving brain functions.

RELATED ART

Cerebral apoplexy (or stroke) refers to a cerebral disease, a mosthighly ranked cause of death in Korea, caused by rupture or obstructionof blood vessels in brain and results in abnormalities in some cerebraltissues. The death rate due to the above disease has been on the risebecause of extended life span due to industrialization and developmentof medical science. Stroke may develop in any part of a body and causedysfunction of the part accordingly. Medically, stoke is divided into‘ischemic stroke’ and ‘hemorrhagic stroke’, and the former, which ismore closely related to hypertension and arteriosclerosis, shows arelatively higher rate of recurrence.

Ischemic stroke is caused by obstruction in from any blood vessel arounda neck (e.g. a carotid artery) to any in brain. As a result, cerebralinfarction occurs and the function of that region may not be recoveredfor good. Therefore, the most important thing in treating stroke isprevention of cerebral ischemia itself together with prevention of riskfactors such as hypertension, diabetes and elevated level of bloodlipid.

Examples of materials currently used for neuronal protection areexcitatory amino acid antagonists such as ganglioside and nimodipine,and GABA agonists such as clomethiazole. Magnesium sulfate and glycineantagonist are under 2^(nd) phase clinical trial, and a large-scaleclinical trial is being performed about piracetam. However, theconventional neuroprotective agents were mainly aimed at acting on eachstep in ischemia development, and thus still remains a need to develop acomposite agent acting on many steps at the same time with little sideeffects and drug complications. Further, functional food, which mayconstantly prevent ischemia and inhibit post-ischemic neuronalapoptosis, has been considered to be more effective than the medicine toprevent ischemic stoke itself.

U.S. Pat. No. 6,245,757 discloses a use of progestin for treating cellimpairment by ischemia. U.S. Pat. No. 6,380,193 discloses apharmaceutical composition comprising poly(adenosine5′-diphospho-ribose)polymerase inhibitor for treating stroke. Also, U.S.Pat. No. 6,288,041 discloses a pharmaceutical composition comprisingsialic acid derivatives for treating stroke.

Parkinson's disease (PD) is one of neuronal degenerative diseases thatmay cause impairment in movement and intelligence and was first reportedby James Parkinson in 1817. In US, the attack rate of this disease isabout 100-150 persons per 100,000 people. The number of current patientsis about 750,000-1,000,000 and about 60,000 new patients are added tothe list each year. Considering the global trend of aging society, itsincidence rate is also expected to increase in Korea. Tissuepathologically, PD induces loss of dopamine neuronal cells in substantianigra and decrease of dopamine in caudate nucleus and putamen, followedby impairment in movement or intelligence such as tremor, bradykinesia,rigidity and disturbance of posture.

Drugs that can supplement functions of dopamine in brain, or prevent ordelay destruction of neuronal cells, or control the accompanying symptomsuch as depression have been used to treat Parkinson's disease. Examplesof those drugs are madopar (levodopa, L-dopa; dopamine precursor),bromidine (dopamine receptor agonist), lisuride, artane(anti-acetylcholine) and cogentin. Of these drugs, levodopa is known tobe most effective intreating Parkinson's disease by supplementingdopamine concentration in brain. However, when administered for morethan 3-5 years, the levodopa shows side effects such as a shortenedeffective time (wearing-off) or large fluctuation in motion controllingfunction (on-off phenomenon) and abnormal motion symptom (diskinesia)(Freed et. al., N. Engl. J. Med. 327:1549-55(1992)).

Further, surgical treatment for Parkinson's disease has been also used,and its examples are thalamotomy, pallidotomy, deep brain stimulationand Neuronal cell transplantation. However, a lasting time of efficacydiffers significantly from patient to patient along with serious sideeffects such as hypophonia accompanying operation, dysarthria, a declinein memory (Ondo et. al., Neurology 50:266-270 (1998); Shannon et. al.,Neurology 50:434-438(1998)).

Treatment of Alzheimer's disease has been recently focused on the factthat Alzheimer's disease may be caused by impaired cholinergic signalingand transmission in cerebral cortex and hippocampus (Bartus et al.,Science. 217(4558): 408-14(1982)); Coyle et al., Science.219(4589):1184-90(1983)). Because this region in brain is associatedwith memory and intelligence, functional defect in this region may causeloss of memory and intelligence. Although the process of impairment inneuronal signaling is still controversial, senile plaque andneurofibrillary tangle (NFT) are considered as main causes. Senileplaque due to the accumulation of amyloid β (Aβ) is a notable feature ofthis disease, and Alzheimer disease may be confirmed by a postmortemexamination (Khachaturian, Arch. Neurol. 42(11):1097-105(1985)).

As a way of treating Alzheimer's disease, a method of increasing ormaintaining acetylcholine amount to inhibit the impairment ofcholinergic signaling or causing acetylcholine to acts more effectivelyon transmission of neuronal cells is provided. Thus, patients ofAlzheimer's disease use compounds for increasing activity ofacetylcholine. The most effect way is to rapidly decompose acetylcholinein synapse, thus inhibiting activity of acetylcholinesterase, and theseinhibitors (e.g. tacrine, donepezil and rivastigmine) are approved byKFDA and currently on market. Despite their effectiveness in preventingfurther destructive progress of the disease, they are not applied torecover patients to pre-illness level.

Some compounds are aimed to improve neuronal condition and maintain agedcells in good function. For example, NGF or estrogen acts asneuroproting agent to delay neurodegeneration and anti-oxidantsdecreases cell damage caused by oxidation of cells. Alzheimer's diseasebecomes serious as amyloid β peptide is accumulated in neuritic space,and amyloid precursor protein (APP) is considered to play a role incombination with proteinase in cells such as α-, β- and γ-secretases.However, the process of amyloid β formation, it is not impossible tocontrol the formation of amyloid β.

It is not certain how the accumulation of amyloid β acts on neuronaltransmission. Abnormally cleaved APP induces amyloid β generation, andplaques are induced by the accumulation of the amyloid β. Thus, variousfactors acting on the cleavage reaction (e.g. inflammation reaction)increase phosphorylation of tau protein, and also increase theaccumulation of paired helical filament (PHF) in combination with NFT,resulting in neurodegeneration and finally expedition of dementia ofAlzheimer's type.

Up to date, the treatment of Alzheimer's disease is just focused onimprovement of the symptom instead of restoring disease process.

U.S. Pat. No. 5,532,219 discloses a pharmaceutical compositioncomprising 4,4′-diaminodiphenylsulfone for treating Alzheimer's disease.U.S. Pat. No. 5,506,097 discloses a pharmaceutical compositioncomprising para-amidinophenylmethanesulfonyl fluoride or Ebelactone Afor treating Alzheimer's disease. U.S. Pat. No. 6,136,861 discloses apharmaceutical composition comprising bicyclo[2.2.1]heptane.

Meanwhile, stress is becoming major problem in health in modern society,and it is reported that, in Korea, ⅓ of the twenties usually experiencemuch stress, and that the women suffer from stress than men in theirteens. Strength of stress depends on personality, interest, means ofrelief from stress, surrounding environment, controlling ability of aperson, and stress is usually followed by depression. Depression mayresults in suicide, and is considered to be a very important disorderbecause of its high rate of occurrence and recurrence. Depression hasbeen reported to be caused by impairment of neurotransmitters such asadrenaline, dopamine or serotonin, and followed by cerebral impairment.Although tricylic antidepression (TAC), especially amitriptyline, is awell known treatment, it has drawbacks of having various side effects.Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) developedin US in 1980's, ranked 7^(th) among 20 international drugs because itovercame the problems of TAC and increase the drug compliance. However,SSRI showed little improvement in efficacy compared with TAC and stillhas serious drug interference.

Further, neuronal disturbance is still induced by stress and there is noway to inhibit the relapse after medicinal treatment of depression, andthus a long-term administration with a lowered dose is only used atpresent. Therefore, it is very important to develop a material withsuperior activity of inhibiting neuronal apoptosis and correctingneuronal transmission system. Furthermore, functional foods withanti-depression activity are also important to be developed, asconsidering a tendency of avoiding visiting treatment institution andoverlooking the induction of disturbance in serotonin neuronal systemand cerebral impairment.

As representative examples of effort to develop medicine for preventingand treating neuronal degenerative disease, U.S. Pat. No. 6,020,127discloses genes encoding proteins suppressing neuronal apoptosis fromhuman chromosome 5q13, and. U.S. Pat. No. 6,288,089 disclose pyridylimidazole for treating neuronal degenerative disease by suppressingapoptosis of dopamine neuron.

SUMMARY

Papers and patents are referred to throughout the present specificationand references are shown in parentheses. The papers and the patents areincorporated by reference herein in their entirety for betterunderstanding the level of related arts and the gist of the presentinvention.

The present inventors have performed an intensive research andexperiments and finally found that silk peptide produced by hydrolysisof silk protein having a certain range of weight average molecularweight has a superior neuroprotective activity such as prevention ortreatment of brain disease and improves various indices for brainfunction, thus completing the present invention.

Therefore, an object of the present invention is to provide a method ofpreparing silk peptide having neuroprotective activity.

Another object of the present invention is to provide a pharmaceuticalcomposition for preventing or treating brain disease.

Still another object of the present invention is to provide a foodcomposition for preventing or treating brain disease.

Another object of the present invention is to provide a pharmaceuticalcomposition for improving brain function.

Still another object of the present invention is to provide a functionalfood composition for improving brain function.

Other objects or advantages of the present invention are betterunderstood by referring to the following Detailed Description andFigures.

DETAILED DESCRIPTION

In one aspect of the present invention, there is provided a method ofpreparing silk peptide with weight average molecular weight of200-100,000 having neuroprotective activity by performing hydrolysis ofsilk protein.

In another aspect of the present invention, there is provided apharmaceutical composition for preventing or treating brain disease,which comprises (a) a pharmaceutically efficacious amount of silkpeptide produced by decomposition of silk protein, and (b) apharmaceutically acceptable carrier.

In still another aspect of the present invention, there is provided afood composition for preventing or treating brain disease, thecomposition comprising silk peptide produced by hydrolysis of silkprotein as an active ingredient.

In a further aspect of the present invention, there is provided apharmaceutical composition for improving brain function, which comprises(a) pharmaceutically efficacious amount of silk peptide produced byhydrolysis of silk protein, (b) a pharmaceutically acceptable carrier.

In a still further aspect of the present invention, there is provided afood composition for improving brain function, which comprises silkpeptide produced by hydrolysis of silk protein as an active ingredient.

Silk protein is used as a staring material in the present invention. Asused herein, the term “Silk” refers to thread produced by silkworm,especially that produced by the larvae of Bombyx mori in forming thecocoons within which the worm is enclosed during the pupa stage.

Silk protein consists of about 75% of fibroin and about 25% of sericin,and the silk fibroin. and silk sericin are used herein as a raw materialof silk peptide. Fibroin protein comprises high amount of glycine andalanine, while sericin has a greater than one third of serine showing atotally different content from that of fibroin. According to recentreport, silk fibroin is a huge protein of 2.3 BG101a consisting ofH-chain (350 kDa), L-chain (26 kDa) and glycoprotein P25 (30 kDa) withmolar ratio 6:6:1, where H-chain forms S—S binding with L-chain and P25is linked to these chains by non-covalent bonds. Due to this structure,silk fibroin behaves like a polymer having alternating layers of acrystallite region and a non-crystallite region.

In a preferred embodiment, the silk protein is silk fibroin.

In a preferred embodiment, the decomposition of silk protein isaccomplished by performing non-limiting reaction selected from the groupconsisting of (i) decomposition in calcium salt solution, (ii) acidhydrolysis, (iii) hydrolysis by an enzyme, and (iv) a combinationthereof. The silk peptide so produced has weight average molecularweight of 200-100,000.

In a more preferred embodiment, the decomposition of silk protein isaccomplished by performing (i) decomposition in calcium salt solution,and sequentially (ii) hydrolysis by protease. Further, the decompositionof silk protein is preferred to comprise steps of (a) dissolving silkfibroin in solution comprising calcium salt, (b) removing the calciumsalt from the solution, (c) adding mixture of flavourzyme and sumizyme,whereby producing silk peptide having weight average molecular weight of200-2,000.

CaCl₂.ethanol is usually used as a calcium salt in the decomposition incalcium salt solution (i). The step (a) is preferred to be performed bydissolving silk fibroin in solution comprising calcium chloride, waterand ethanol at 60-95° C., more preferably 70-95° C., most preferably85-90° C. The step (b) may be performed by using various conventionalmethods such as gel filtration chromatography. The step (d) is preferredto be performed at 45-60° C., more preferably 50-60° C., most preferablyat about 55° C. Silk peptide so produced has a relatively high weightaverage molecular weight of 25,000-50,000, and is not appropriate forfood and medication due to its low body's absorption while it is stilluseful for cosmetics. Silk peptide produced by (ii) acid hydrolysis hasweight average molecular weight of 200-10,000. While various acids maybe used, a strong inorganic acid is preferred, more preferablyhydrochloric acid. The temperature of the reaction (ii) is preferred tobe 70-120° C., more preferably 90-110° C. and most preferably 110° C.The reaction (ii) has an advantage that a low molecular weight ofpeptide is obtained, while it is difficult to appropriately control therange of molecular weight. In one embodiment, the acid hydrolysis (ii)comprises steps of (a) performing hydrolysis in hydrochloric acidsolution at 70-120° C., (b) raising pH by adding alkaline solution tothe hydrolysis solution, and (c) removing a salt produced in the step(b), thereby producing silk peptide having weight average molecularweight of 200-3,000. Most preferably, silk peptide so produced hasweight average molecular weight of 200-1,500. Most appropriate alkalinesolution used in the step (b) is sodium hydroxide solution. Variousknown methods such as gel permeation chromatography may be used as thestep (c). Further, peptide produced by acid hydrolysis has substantially100% solubility in water while it shows very low solubility in organicsolvent such as ethanol, methanol, acetone and dimethylformamide.

The aforementioned hydrolysis by an enzyme is performed by usingprotease. Preferably, the hydrolysis by protease is performed randomlyby using a sequence-non-specific protease. The representative example ofthe protease includes but is not limited to trypsin, pepsin, alcalase,thermoase, flavourzyme, sumizyme, protamex and protin. In a preferredembodiment, the enzyme used herein is thermoase, flavourzyme, sumizymeor a mixture thereof.

In the case of (iv) the combined reactions, the decomposition of silkfibroin is accomplished by sequentially performing (i) decomposition bycalcium salt or hydrolysis by a weak acid or a base and (ii) hydrolysisby an enzyme. Preferably, the decomposition of silk fibroin isaccomplished by sequentially performing (i) decomposition by a calciumsalt, and (ii) hydrolysis by thermoase, flavourzyme, sumizyme, and amixture thereof. The silk peptide so produced has weight averagemolecular weight of 200-15,000, preferably 200-4,000, more preferably200-2,000, and most preferably 200-1,200. The silk peptide hassubstantially 100% solubility in water and shows very low solubility inan organic solvent such as ethanol, methanol, acetone and dimethylamide.In a most preferred embodiment, the decomposition of silk fibroincomprises steps of (a) dissolving silk fibroin in solution comprisingcalcium salt, (b) removing the calcium salt from the solution, (c)adding a mixture of flavourzyme and sumizyme.

Preferred peptide herein is (a) one prepared by hydrolysis using silkfibroin as substrate; and (b) one prepared using silk fibroin assubstrate by sequentially performing decomposition by calcium salt anddecomposition by an enzyme selected from the group consisting of trysin,pepsin, alcalase, thermoase, flavourzyme, sumizyme and a mixturethereof. More preferred peptide is (c) one prepared using silk fibroinas substrate by performing decomposition by calcium salt andsequentially decomposition by an enzyme selected from the groupconsisting of thermoase, flavourzyme, sumizyme and a mixture thereof.

A composition comprising silk peptide herein has an activity inpreventing or treating brain disease mainly due to its neuroprotectiveactivity (i.e. protective activity for neuronal cell). As used herein,the term “neuronal cell” includes central nervous system, brain,brainstem, spinal cord, neuron having a structure connecting centralnervous system and peripheral nervous system, and neuronal supportingcell, Glia and Schwann cell. As used herein, the term “neuroprotectiveactivity” or “protective activity for neuronal cell” refers to theeffect of reducing or ameliorating nervous insult, and protecting orreviving nervous cell that has suffered nervous insult. As used, herein,the term “nervous insult” refers to any damage to neuronal cell ortissue resulting from various causes such as metabolic, toxic,neurotoxic and chemical causes.

Representative examples of disorders, which the composition herein maybe applied to, include without limitation neurodegenerative disorder,ischemia-reperfusion injury and mental disorder, and more specificallyneurodegenerative disorder such as Alzheimer's disease, Huntington'sdisease, Parkinson's disease and amyotrophic lateral sclerosis,ischemia-reperfusion injury such as stroke (especially ischemic stroke)and mental disorder such as depression, schizophrenia and post traumaticstress disorder. The composition herein is especially useful forpreventing or treating disease due to neuronal damage related toischemia or reperfusion such as stroke.

The aforementioned effect of the composition herein is mainly due to itsneuroprotective activity. The neuroprotective activity may beaccomplished via various mechanisms such as inhibition of neuronal celldeath, which includes necrosis and apoptosis of neuronal cell. Theinhibition of neuronal apoptosis may be accomplished by inhibitingactivity of caspase (Guy et. al., Cell 91:443-446(1987)), one of targetsof silk peptide herein.

The silk peptide herein also has a superior activity for improving brainfunction or cognitive function. As described in Examples, silk peptideherein remarkably raises various indices in support for improvement ofbrain function such as memory quotient (‘MQ’), learning slope, memoryretentiveness, recall efficiency, drawing/memory consensus,language/view consensus, intelligence/memory consensus, short termmemory, and attentive concentration.

Silk peptide herein also has a superior activity for improving orpreventing damaged brain function due to the brain disease, especiallyby suppressing the decrease of acetylcholine. Further, the silk peptideherein prevents the damage of brain function by inhibiting neuronalapoptosis. In a preferred embodiment, the brain function is learningcapability and/or retentive faculty.

Acetylcholine is neurotransmitter that is projected from basal gangliato cerebral cortex or hippocampus, thereby performing a very importantactivity for normal brain function (Richter et. al., Life Sci.19;26(20)1683-9(1980)). Especially, learning and memory has been knownto be varied by drug acting on acetylcholine signaling. Investigation ofpeople who died of Alzheimer type dementia has showed that the majoracetylcholinergic neurons are much damaged. Choline agonists and cholineesterase inhibitors has been used for patients, as it is recently knownthat the increase of acetylcholine acts in treating or preventingdementia by improving cognitive function and inhibit the development ofdementia. Up to present, there have been developed acetylcholineprecursor such as Lecithin; receptor agonist such as RS-86, nicotine andacetylcholine esterase inhibitor such as Tacrine and Aricept, the formerof which was approved by KFDA and is on the Korean market and the latterof which was also recently approved by KFDA. However, their use is stillopen to argument because their effects do not last long, are weak andalso seriously toxic, in contrast, silk peptide herein shows negligibletoxicity to human body, thus being very useful in medication or food forimproving brain function.

As used herein, the term “prevention” or “preventing” refers toinhibiting the generation of disorders or diseases in animal includinghumans who are not diagnosed to have but are susceptible to suchdisorders or diseases. As used herein, the term “treatment” or“treating” refers to (a) inhibiting the development of disorders ordiseases; or (b) ameliorating or (c) removing the disorders or diseases.

Any pharmaceutically acceptable carriers may be used herein and theirrepresentative examples include without limitation carbohydrates (e.g.lactose, amylase, dextrose, sucrose, sorbitol, mannitol, starch andcellulose), acacia rubber, calcium phosphate, alginate, gelatine,calcium silicate, fine crystallite cellulose, polyvinylpyrrolidine,cellulose, water, syrup, salt solution, alcohol, Arabian rubber,vegetable oil (e.g. corn oil, cotton seed oil, soybean oil, olive oiland coconut oil), poly(ethylene glycol), methyl cellulose, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineraloil. Pharmaceutical composition herein may further include withoutlimitation a lubricant, a wetting agent, a sweetening agent, flavors, anemulsifying agent, a suspending agent and a stabilizer.

The pharmaceutical composition herein may be orally or parenterallyadministered, and examples of parenteral administration includeintravenous, subcutaneous and intramuscular injection.

Appropriate dosage level of the pharmaceutical composition herein may bedetermined by considering various information such as formulationmethod, administration type, age, body weight, sex, physical conditionsfood, administration time and route, excretion and reaction sensitivity.Physicians with average skill may easily determine and diagnose dosagelevel of medicine effective for treating or preventing target disordersor diseases. In a preferred embodiment, the dosage level for an adult isonce-a-day administration and 0.05-10 g per dose.

The pharmaceutical composition may be prepared in unit dosage form or inmultidose container by using pharmaceutically acceptable carriers orfillers according to the conventional method. Representative examples offormulation type include oily or aqueous solution, suspension, emulsion,extract, powder, granule, tablet and capsule, and the formulation mayfurther comprise a dispersing agent or a stabilizer.

Meanwhile, a food composition herein may comprise conventional additivessuch as protein, carbohydrate, fat, nutrient, and flavor. For example,liquid medicine as an embodiment of the present invention may furthercomprise citric acid, liquid fructose, sweet, glucose, acetic acid,malic acid, fruit syrup, eucommia bark extract, jujube extract, andglycyrrhiza extract. Considering easy accessibility of food, the foodcomposition herein is very useful in preventing or treating braindisease or oxidative-stress-induced disorders and in improving brainfunction.

In addition to the aforementioned various activities, the compositionherein shows very low side effect to human body compared to medicineprepared by chemical synthesis because the composition herein comprisesnatural silk peptide as an active ingredient.

The present invention is described more specifically by the followingExamples. Examples herein are meant only to illustrate the presentinvention, but in no way to limit the scope of the claimed invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the result of MIT reduction experimentation toverify the effect of a kind of silk peptide herein on neuronalapoptosis.

FIG. 2 is a graph showing the result of MIT reduction experimentation toverify the effect of another kind of silk peptide herein on neuronalapoptosis.

FIG. 3 is the result of Hoechst staining experiment and shows that akind of silk peptide herein inhibits neuronal apoptosis.

FIG. 4 is the result of Hoechst staining experiment and shows thatanother kind of silk peptide herein inhibits neuronal apoptosis.

FIG. 5 is a graph showing that a kind of silk peptide herein inhibitsthe activity of caspase.

FIG. 6 is a graph showing that another kind of silk peptide hereininhibits the activity of caspase.

FIG. 7 is a photograph showing that a kind of silk peptide hereininhibits the generation of reactive oxygen in cells.

FIG. 8 is a photograph showing that another kind of silk peptide hereininhibits the generation of reactive oxygen in cells.

FIG. 9 is a photograph showing the effect of silk peptide herein on theparts of ischemic cerebral infarction.

FIG. 10 is a graph showing the effect of silk peptide herein on parts ofischemic cerebral infarction.

FIG. 11 is a graph showing the effect of silk peptide herein on parts ofischemic cerebral infarction.

FIG. 12 is a graph showing the effect of silk peptide herein on parts ofischemic cerebral infarction.

FIG. 13 is a result of passive avoidance test showing that silk peptidehere prevents the cerebral damage due to ischemic-reperfusion.

FIG. 14 is a result of 8-arm radial maze test showing that silk peptidehere prevents the cerebral damage due to ischemic-reperfusion.

FIG. 15 is a result of H&E dyeing of hippocampus showing that silkpeptide here prevents the cerebral damage due to ischemic-reperfusion.

FIG. 16 is a result of unilateral rotation response due to apomorphineby using animal model with Parkinson's disease.

FIG. 17 is a graph showing that silk peptide herein has aneuroprotective activity on dopamine neuronal cell.

FIG. 18 is a malondialdehyde experiment result showing that silk peptideherein has a remedial efficacy on Parkinson's disease due to its ananti-oxidative function.

FIG. 19 is a result of tyrosine hydroxylase dyeing experiment showingthat silk peptide has a remedial efficacy on Parkinson's disease.

FIG. 20 is a result of tyrosine hydroxylase dyeing experiment showingthat silk peptide has a remedial efficacy on Parkinson's disease.

FIG. 21 is a graph showing that silk peptide herein inhibits thedecrease in concentration of cerebral acetylcholine.

FIG. 22 is a graph showing that silk peptide has an anti-depressionactivity when administered one time.

FIG. 23 is a graph showing that silk peptide has an anti-depressionactivity when administered repeatedly for a long time.

FIGS. 24 and 25 are graphs showing that BF-7, a capsule comprising silkpeptide herein has an effect of increasing memory quotient (MQ).

FIG. 26 is a graph showing BF-7, a capsule comprising silk peptideherein has an effect of improving recall efficiency.

FIG. 27 is a graph showing BF-7, a capsule comprising silk peptideherein has an effect of improving drawing/memory consensus.

FIG. 28 is a graph showing BF-7, a capsule comprising silk peptideherein has an effect of improving intelligence/memory consensus.

FIG. 29 is a graph showing BF-7, a capsule comprising silk peptideherein has an effect of improving intelligence/memory consensus.

EXAMPLES Example I Preparation of Functional Silk Peptide BG201 andBG101 Example I-1 Preparing Pure Silk Fibroin

Silk protein used herein was prepared by feeding allaged mulberry leavesto Bombyx mori. Pupae were removed from cocoons to get rid of sericin.150 g of cocoons were added into mixture of 8 L of water, 0.45 g ofsodium carbonate and 0.75 g of marseillous soap, and the mixture allowedto be boiled for 40 minutes and, this step was repeated twice. Themixture was boiled for 20 minutes by using appropriate amount of water,repeated twice more and washed three times and dried, thus allowingsericin to be solved off. The reduced mass was 37 g (25% reduced), andpure silk fibroin was prepared by washing and drying the mixture.

Example I-2 Preparation of Silk Peptide BG101

35 g of fibroin prepared in Example I-1 was added into solutioncomprising calcium chloride (CaCl₂, 1^(st) grade, Mw=110.99), H₂O andethanol with a molar ratio of 1:8:2, and dissolved at 90° C. for 5hours. The mixture was filtered to remove impurities by using gauze andnon-woven fabric, and diluted by adding the same amount of distilledwater. Neutral salt was removed by using gel chromatography device with10 cm of diameter and 1 m of length (GradiFac system, Pharmacia Biotech,Sephadex G-25 Media, HiLoad P-50 Pump UV-1 Monitor, Sweden). To theprepared fibroin solution, 1% of one out of the following combination ofproteases were added and hydrolysis was performed at 55° C. for 5 hours:(1) 1:1 of flavourzyme and sumizyme (NOVA, US), (2) 1:1 of trypsin andpepsin, (3) 1:1 of thermoase alcalase. The mixture was thermally treatedat 100° C. for 5-10 minutes to remove the activity of enzyme, and cooleddried, thus obtaining powders named “BG101”.

Example I-3 Preparation of Silk Peptide BG201

113 g of fibroin prepared in Example I-1 was added to 3,400 mL ofchloric acid solution (25%), and hydrolysis was performed at 110° C. for12 hours, followed by addition of sodium hydroxide solution (4 M) tomaintain pH to 5.0-5.5. The solution was paper-filtered and passedthrough filter filled with activated carbon, followed by electrolyticdesalting process by using an electro-dialysis device that automaticallycontrol current and voltage to 20 mA and 15 V, respectively. Powderswere prepared by using a spray drier and named as “BG201”.

Example II Analysis of Silk Peptide Example II-1 Molecular Weight

Absolute molecular weights of BG101 and BG201 were determined by usinggel filtration chromatography method. Particularly, the concentration ofthe sample was controlled within 0.5% by using 0.2 N NaNO₃ solution asbuffer solution, and property data of the sample were obtained such asrefractive index (RI), light scattering (LS), diffraction pressure, UVadsorption (280 nm). GPC system (Viscotec, US) was used thatautomatically calculates the absolute molecular weight distribution fromthe data. As a standard, polyethylene oxide (PEO, M_(w)=110,000) wasused for verifying reproducibility, and weight average molecular weightsof BG101 and BG201 were verified as 1070 and 850, respectively.

Example II-2 Solubility in Various Solvents

0.1 g of silk peptide powders were dissolved in 10 mL of distilledwater, ethanol, methanol, acetone and dimethyl formamide andsolubilities (%) of BG101 and BG201 in those solvents were measured, asshown in Table 1. BG101 and BG201 were verified to be fully soluble indistilled water while the solubilities in organic solvents such asethanol and methanol were drastically decreased.

TABLE 1 Distilled Dimethyl Solvents water Ethanol Methanol Acetoneformamide BG101 100 20 45 30 50 BG201 100 30 50 32 50

Example II-3 Solubility at Carious pH Values

0.1 g of silk peptide powders were dissolved in 10 mL of distilledwater. Solubilities at pH 3, 5, 7, 9 and 11 were measured and pH wascontrolled by using 0.1 N of hydrochloric acid and sodium hydroxide. Asshown in Table 2, silk peptide was verified to be fully solubleirrespectively of pH value of solvents.

TABLE 2 pH 3 5 7 9 11 BG101 100 100 100 100 100 BG201 100 100 100 100100

Example II-4 Amounts of Amino Acids

Compositional analysis was performed to determine the amounts of aminoacids in BG101 and BG201, respectively. 0.05% of each sample was addedin 1 mL of chloric acid solution and nitrogen treatment was performed,followed by hydrolysis for 18 hours at 110° C. The solution was totallyevaporated off hydrochloric acid and diluted with loading buffersolution of pH 2.2. Compositional analysis on amino acid was performedby using automatic amino acid device (Biochrom 20 Plus, Sweden) and theresults were provided in Table 3.

TABLE 3 Amino acid BG201 BG101 Gly 43.2 42.46 Ala 27.2 26.64 Ser 15.9411.24 Tyr 2 4.41 Val — 2.02 Asp 1.45 2.11 Glu 1.61 1.68 Thr 0.6 — Met0.15 0.11 Ile 0.98 0.65 Leu 0.71 0.57 Phe 1 0.76 His 0.52 0.52 Lys 0.381.04 Arg 1.01 1.04 Total 96.21 95.25

As shown in Table 3 above, BG101 and BG201 are similar to each other inthe contents of amino acids, while they showed noticeable difference intheir in vivo or in vitro activities, which means that the resultantsilk peptides have different structures depending on their preparationmethods.

Experimental Example I Verification of Neuroprotective Activity andAnti-Oxidative Function

Experiments set forth below were performed to verify the effect of silkpeptide on neuronal cells.

Experimental Example I-1 Selection of Neuronal Cells and Culture ofCells

Pure neuronal cells were prepared by performing primary culture ofneuronal cells according to Okuda's method (Okuda S. et al.,Neuroscience 63(3):691-9(1994). Particularly, striatum was prepared from15 day embryo of a mouse (E15) and each cell was separated by treatmentof 0.25% trypsin and 0.01% DNase I, and further separated with pipette.Each cell was placed on PEI coating plate at a density of 1×10⁵cells/cm², and culture of neuroglial cell was inhibited by using ara-C.Further, neuron-derived cell lines, SK-N-SH or SHS-Y5Y (ATCC, US) wereplaced on PEI-coating plate at a density of 80% and culture by usingculture solution comprising DMEM or RPMI and 10% of FBS. As set forth inthe following Experimental Examples, pretreatment was performed by usinglow-serum medium (1% FBS contained) before treatment of amyloid β(Aβ),6-hydroxydopamine (6-OHDA), ceramide, H₂O₂ or 3-hydroxykynurenine(3-HK).

Experimental Example I-2 MIT Reduction Experiments (Verification of CellSurvival)

To verify the effect of BG101 and BG201 on apoptosis of neuronal cells,MTT reduction experiment was performed by modifying a known method(Shearman et al., Proc. Natl. Acad. Sci. 91(4):1470-4(1994), Shearman etal., J. Neurochem. 65(1):218-27(1995), and Kaneko et al., J. Neurochem.65(6)2585-93(1995)) as follws. Neuronal cells, which were selected andcultivated in the Experimental Example above, were treated with 10 pMBG101 or BG201 for 6 hours with, followed by addition of 10 μM Aβ, 10 μM6-OHDA, 10 μM ceramide, 300 μM H₂O₂, or 250 μM 3-HK. The added materialsinduced about 50% apoptosis within 36 hours due to the cellularcytotoxicity.

The samples were incubated in 5% CO₂ at 37° C. for 48 hours, followed byfurther culture for 4.5 hours after addition of3-(4,5dimeylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT: Sigma,US) solution to the concentration of 0.5 mg/mL.

Formazan precipitates formed by MTT reduction were dissolved in thesolution (0.1 N HCl in anhydrous isopropanol), and absorbance at 570 nmwas determined by using ELISA Reader (Molecular Devices, US). As shownin FIGS. 1 & 2, a value of each sample was determined by selectingappropriate value between cell viability of 100% (control group, onlysolvent used) and cell viability of 0% (cells are totally destroyed by0.9% of Triton X-100. The values in FIGS. 1 & 2 are expressed as‘mean±standard deviation’. BG101 and BG201 were verified to be effectivein inhibiting neuronal apoptosis (P<0.05).

Meanwhile, to check the aforementioned results, cell viability wasfurther determined as ‘surviving cells/total cells’ by staining withtryphan blue or crystal violet to measure the each number of total cellsand apoptotic cells with hematocytometer. The obtained results wereverified to be very similar to the aforementioned results. Further, itwas verified that cell death occurs in the form of apoptosis, which wasalso confirmed by the observation of DNA degradation after Hoechststaining.

Experimental Example I-3 Hoechst Staining

As set forth in Experimental Example I-2, neuronal cells were treatedwith BG101 or BG201 for 6 hours with and cultivated, followed byaddition of 10 μM Aβ, 10 μM 6-OHDA, 10 μM ceramide, 300 μM H₂O₂, or 250μM 3-HK. The cells were fixed with 4% paraformaldehyde (in PBS, pH 7.4)for 15 min and washed with BPS, followed by staining with 8 μg/ml ofHoechst dye 33258 solution (Sigma, US) for 5 min. They were washed twicewith distilled water and mounted with glycerol:PBS mixture (9:1), andanalyzed under fluorescent microscopy (Olympus microscope, Japan), asshown in FIGS. 3 & 4. The cells, which were treated only with apoptosisinducing agent, showed a typical apoptosis morphology such as chromatincondensation and nuclear fragmentation, which was effectively inhibitedby pretreatment of BG101. and BG201 followed by insult treatment. Mockreferring to control group shows normal nuclear morphology. As shown inFIG. 2, BG101 and BG201 herein were verified to be effective insuppressing apoptosis, which confirms the reliability of theaforementioned Experimental Example I-2.

Experimental Example I-4 Caspase Activity Experiment

The following experiments were performed to verify that the inhibitionof apoptosis was due to the inhibition of caspase activity. As set forthin Experimental Example I-2, neuronal cells were treated with BG101 orBG201 for 6 hours with and incubated, followed by addition of 10 μM Aβ,10 μM 6-OHDA, 10 μM ceramide, 300 μM H₂O₂, or 250 μM 3-HK.Cell-dissolved solution was obtained by destroying 10×10⁶ cells per P100plate with cell-dissolving buffer solution (50 mM Tris-Hcl, 0.03%IGEPAL, 1 mM DTT, pH 7.5). 500 μM of Ac-DEVD-AMC (Enzyme SystemsProducts, Canada), fluorescence producing substrate of caspase, whichwas prepared from HEPES buffer solution (40 mM HEPES, pH 7.5, 20%glycerol, 4 mM DTT) was added in 50 μL of the cell-dissolved solution,and reaction was performed at 37° C. for 1 hour. A positive control wasprepared by pretreating 10 μM of zVAD-FMK (Enzyme Systems Products, ESP,Canada), pan-caspase inhibitor. The degree of fluorescence that wasemitted when the substrate was cleavaged by caspase was measured byusing fluorescence analyzer (Perkin-Elmer Luminometer; excitationwavelength 380 nm, emission wavelength 420-460 nm). To quantify themeasured fluorescence, standard curve was prepared by using degree offluorescence of cleavaged FMK (FIGS. 5 & 6; P<0.05). As shown in FIGS. 5& 6, BG101 or BG201 herein is verified to inhibit the caspase activitythat is caused by an apoptosis-inducing agent. Thus, it can be concludedthat apoptosis inhibition effect of BG101 and BG201 is related to theinhibition of caspase activ

Experimental Example I-5 Quantitative Determination of Reactive Oxygenwithin Cells

Reactive oxygen is a main reason of aging and also directly orindirectly causes many kinds of disease. Thus, the present inventioninvestigates the effect of silk peptide herein on the reactive oxygen.

As set forth in Experimental Example I-2, cells were treated with BG101or BG201 for 6 hours with and cultivated, followed by addition of 10 μMAβ, 10 μM 6-OHDA, 10 μM ceramide, 300 μM H₂O₂, or 250 μM FeSO₄ or 3-HK.The cultivated cells were treated with 10 μM

DCFDA (6-carboxy-2′,7′-dichloro-dihydrofluoresceine diacetate,dicarboxym-ethylester) dissolved in HCSS buffer solution (20 mM HEPES,2.3 mM CaCl₂, 120 mM NaCl, 10 mM NaOH, 5 mM KCl, 1.6 mM MgCl₂, 15 mMglucose) and 2% of pluronic F-127, suspension supplementer, at 37° C.for 30 minutes. DCF fluorescence by reactive oxygen in cells wasobserved at room temperature by using Olympus IX70 equipped with mercurylamp fluorescence attachment (excitation wavelength 488 nm, emissionwavelength 510 nm), and the screen was captured by CCD camera andanalyzed by using NIH Image 1.65 program (FIGS. 7 & 8).

Mock in FIGS. 7 & 8 refer to specimen treated only with a solvent.Reactive oxygen was verified to generate by treatement of Aβ, 6-OHDA,ceramide, H₂O₂, FeSO₄ or 3-HK (FIG. 4), while the pretreatment of BG101or BG201 remarkably inhibited the reactive oxygen generated by anapoptosis-inducing agent.

Therefore, it can be concluded that BG101 or ED has an anti-oxidativeactivity by inhibiting generation of reactive oxygen induced by amyloidβ, 6-OHDA, ceramide, H₂O₂, FeSO₄ or 3-HK.

Experimental Example II Ischemic Animal Model Experiment

The following experiments were performed to investigate the effect ofsilk peptide BG101 or BG201 herein on the area of ischemic infaction.

Experimental Example II-1 Local Ischemia Induced Animal Model A.Medicine Treatment and Preparation of Local Ischemia Induced AnimalModel

Local ischemia induced, animal model was prepared by using maleSprague-Dawley rat (200-250 g). Vehicle treated group (n=6) were orallyadministered with 1 g/Kg of BG101 1 hour before or after ischemiainduction, and vehicle control group (n=6) were administered with thesame amount of a saline solution. The test subject animals wereadministered with 30-40 mg/Kg of ketamine by intramuscular injection andanesthetized. Common carotid artery, external carotid artery andinternal carotid artery were separated. Superior parathyroid gland andposterior fossa, which are branches of the external carotid artery, andpterygopalatine, which is a branch of the internal carotid artery, wereelectrocauterized, and the external carotid artery were cut. Occlusionof origin of middle cerebral artery was performed by incorporating 4-0nylon thread (ETHICON, INC, US) into the internal carotid artery throughthe external carotid artery, and placing 10-48 mm of nylon thread withinthe common carotid artery.

B. Decrease in Area of Ischemic Infarction

Brains were dissected from the animal 6 hours after induction ofischemia. The dissected brains were cut at interval of 2 mm fromanterior pole, and reacted with 2% triphenyl tetrazolium chloride (TTC,Sigma, US) at 37° C. for 30 minutes, followed by fixing using 4%paraformaldehyde (Sigma, US). Tissue parts were photographed (FIGS. 9 &12) and areas of red-stained normal region and white infarction regionswere measured by using MCID image processing system (Imaging ResearchInc., Canada), thereby calculating the average ratio of the areas (FIG.10).

As shown in FIG. 9, silk peptide herein decreases the area of infarctionregion to the level that statistical significance might be acknowledged(P<0.05). The effect of decreased infarction was found throughout thebrain (FIG. 10). Further, the volume ratio of infarction region to totalbrain was also verified to be decreased by pretreatment of BG1-1, as setforth in FIGS. 10 & 11 (average±standard deviation).

Furthermore, as illustrated in FIG. 12, the region where ischemicinfarction was also verified to be decreased when silk peptide hereinwas treated after the ischemic infarction, was induced.

Therefore, silk peptide was verified to have activity of preventing andtreating ischemic stroke and be useful in functional food or drugs ofthat purpose.

Experimental Example II-2 Local Ischemic Reperfusion Induced AnimalModel A. Medicine Treatment and Preparation of Local IschemicReperfusion Induced Animal Model

Male Sprague-Dawley rat (200-250 g) was administered with 30-40 mg/kg ofketamine by intramuscular injection, and anaesthetized. Common carotidartery, external carotid artery and internal carotid artery wereseparated. Superior parathyroid gland and posterior fossa, which arebranches of the external carotid artery, and pterygopalatine, which isbranch of the internal carotid artery, were electrocauterized, and theexternal carotid artery were cut. 4-0 nylon thread was incorporated intothe internal carotid artery through the external carotid artery and10-18 mm of nylon thread was placed within the common carotid artery.Reperfusion was performed by suturing the cut skin and removing thenylon thread 1 hour after recovery from anaesthesia.

1 g/kg of BG101 or BG201 was administered once a day for 7 days from thenext day of ischemnia-reperfusion. Ischemia control group, administeredwith the same amount and frequency of a physiological saline solutionwith normal. control group and drug treated group, was used as vehiclecontrol group. Passive avoidance test and 8-arm maze test were performed7 days after induction of ischemia-reperfusion.

B. Analysis of Learning and Memory B-1. Passive Avoidance Test

Automated shuttle box (Model PACS-30, Columbus Instruments InternationalCoiupany) was used as test device. The shuttle box was divided into tworooms with the same area (19″L×9″W×10.875″H) by middle door(3″L×2.625″W), and their floors were equipped with current-generatingdevice. Each room might be lighted a 20 W light bulb on hingedplexiglass lid. A white rat might enter a dark room through the door.Noise was control below 60 dB and the test was performed in the darkroom. The Dawley rat was initially placed in a lighted room and moved toa dark room when the door was opened. At this time, the door wasautomatically closed and light was turned off. This test was repeateduntil the rat moved to the dark room within 20 seconds. 26 hours afterthe end of this discipline, when the rat enter the dark room, the doorwas closed and 1 mA of current was generated on a floor of the dark roomfor 3 seconds. 7 days after induction of local ischemia-reperfusion,this rat was placed in the lighted room, and time required for the ratto move to the dark room was measured. The time was limited to 5 minutes(FIG. 13).

As shown in FIG. 13, latency time is related to impairment and recoveryof memory, and the elongation of the latency time means improvement ofmemory. The latency time was not varied with sham operated controlgroup, while it was significantly decreased with ischemia control groupwhich was administered with solvent only (p<0.05) Meanwhile, latencytime was verified to be recovered significantly into normal condition incase of BG101 or BG201 treated group (P<0.05). Especially, BG101 wasfound to be more efficacious because it caused more effective recoveryof latency time than BG101.

B-2. 8-arm Radial Maze Test

8-arm radial maze (Etho Vision, Netherlands) was used as a test device.It was 45 cm above from the bottom, wherein eight arms (60 cm of lengthand 12 cm of width) are protruded out of the octagonal center(radius: 34cm) and the arms and the central part consist of walls (45 cm height).Feed cup was placed on end of each arm. Sunflower seeds were used asreward. Darkness was maintained around the maze by using a 50 W lightbulb, and the maze was monitored with video camera. Learning test wasperformed after decreasing feeds to 80% for 1 week. Male rat was left inmaze 3 times a day for 3 days for discipline, and frequency and time ofvisiting arms were measured until the rat ate all the seeds. Thediscipline was continued until the number of error reached below 2 timeswithin 2 minutes. Entering the already-visited arms again was consideredas an error.

Ischemia animal model was prepared by using the disciplined female ratas follows. The rat was administered with 1 g/kg of BG101 or BG201 oncea day for 7 days, and the number of error and latency time was comparedto those of control group.

8-arm maze test was performed by using ischemia animal model prepared bystopping, middle cerebral artery (MCA), to verify the activity of BG101or BG201 on spatial memory that cannot be confirmed by passive avoidancetest. Test was performed for 5 days from a week after administration,and the control group did not show any improvement between before andafter operation (FIG. 14). In the case of BG101 or BG201 treated group,the number of error was significantly reduced from the third day, andespecially BG101 treated group show larger decrease. Until the last day,BG101 or BG201 treated group maintained lowered number of error, whilethe control group treated with only solvent did not show any recovery(*, P<0.05).

The aforementioned results show that temporary obstruction of middlecerebral artery impaired spatial memory, and repeated administration ofBG101 or BG201 recovered the impairment of learning and memory abilitydue to ischemia. Measured values in FIG. 14 refer to mean ±standarddeviation (SD).

C. Staining of Hematoxylin & Eosin (H&E) in Hippocampus

It has been reported that deterioration of memory and learning abilitydue to cerebral ischemia is related to impairment of hippocampus (Hodgeset al., Neuroscience, 72(4), 959-88, 1996). Thus, activity of thepresent invention was measured in certain regions of hippocampus (CA1,CA2, DG).

C-1. Preparation of Tissue

Test animal experiencing the Experimental Example II-2B wasanaesthetized with 400 mg/mL of chloral hydrate, and 200 mL of 0.1 Mphosphoric acid buffer solution (PBS, pH 7.4) was perfused throughheart, thus removing blood component in blood vessel, followed byreperfusion of 250-300 mL of fixing solution (4% paraformaldehyde/PBS).Brain was dissected and post-fixed with the fixing solution at 4° C. for15-24 hours. After washing the fixing solution with PBS, 10%, 20% and30% sucrose solutions were incorporated in this order, thus preventingice crystal. Brain tissue was embedded with embedding solution andquick-frozen in isopentane prefrozen with liquid nitrogen. Thecontinuous coronal slicing of brain tissue was performed into 10 μmthickness by using microtoming device (Cryostat; Reichert Frigocut model2000), immediately attached to slide glass coated with gelatine, driedat room temperature for 1 hour, and placed at −70° C.

C-2. H&E Staining

The slide glass was washed with distilled water and incubated withhematoxylin for 10 minutes. After the reaction, the slide glass wastreated with 70% ethanol comprising 1% of HCl, thus removing remnanthematoxylin, and fixed by treating ammonia. The tissue was stained witheosin for 20 seconds and washed with distilled water. It was dried with70%, 90% and 100% ethanol, respectively, and treated with xylene andsealed with covering glass and Canadian balsam, followed by observationwith optical microscope.

C-3. Analyzing Result of H&E Staining

As a result of H&E staining in hippocampus with BG101, in the case ofcontrol group induced with ischemia only, eosinophilic phenomenon wasfound throughout the hippocampus (CA1, CA2, DG), which is typical withcell apoptosis (FIG. 15, arrow). Meanwhile, BG101 treated group showed asignificant increase of normal cells.

Although exact mechanism remains to be further studied, BG101 or BG201is verified to have activity of preventing neuronal apoptosis andcerebral impairment, and be useful in new medicine for treatingischemia.

Experimental Example III Experiment Using Parkinsonism Animal Model

6-hydroxydopamine (6-OHDA), which is widely used for inducingParkinson's disease, is absorbed through catecholaminergic neuronal cellmembrane and, shows a selective toxic effect on catecholaminergicneuronal cell. Animal model, which was impaired in dopamine neuronalcell by incorporating 6-OHDA in one side brain, is widely used becausethe other side brain may be used as control group, thus enabling tocompare between spontaneous motion and drug-induced rotational motion.

Following experiments were performed to verify the effect of BG101 andBG201 on Parkinson's disease.

Experimental Example III-1 Preparation of Progressive ParkinsonismAnimal Model

Animal model was prepared by incorporating 6-OHDA in one side striatumof rat and inducing gradual regeneration in dopamine neuronal cellsaccording to Joo's method (Joo W S et al., Neuroreport, 9(18),4123-4126, 1998) as follows.

Male Sprague-Dawley rats, 8 weeks old (around 200-250 g), which werepurchased from Dae Han BioLink Co., Korea), were administered with 3mL/kg of equithesin by intraperitoneal injection. Skulls of theanaesthetized animals were perforated by using stereotaxic frame (DavidKopf, US), and shame group was injected with 0.2 mg/mL of ascorbinicacid in their right corpus striatum by using Hamilton syringe (10 μL,26G needle) at 1 μL/min, while leisioned group and BG101 or BG210treated group were injected with dopamine hydroxide (20 μg/5 μL freebase in 0.2 mg/mL ascorbinic acid) in the same manner (Paxinos et al.,J. Neurosci. Methods. 3(2), 129-149, 1980). 5 minutes after drug wasinjected, the need was withdrawn at a rate of 1 mm/min, and the incisionparts were sutured. Meanwhile, BG101 or BG201 was orally administered intwo doses of 1 g/kg and 5 g/kg.

In a progressive parkinsonism animal model, the effect of dopaminehydroxide was first confirmed by analyzing behavioral change usingapomorphine 14 days after lesion was prepared, and their brains, whichwere extracted after putting cerbical vertebral out of joint the nextday, were used in the experiments. To verify protective effect of BG101or BG201 on dopamine neuronal cells, lipid peroxidation level, dopamineconcentration in corpus striatum by using HPLC and immunohistochemicalstaining on tyrosine hydroxylase were performed.

Experimental Example III-2 Unilateral Rotational Response Induced byApomorphine

To verify behavioral change with time in parkinsonism animal modelprepared by using dopamine hydroxide, 15 mg/kg of apomorphine wassubcutaneously injected in posterior neck 14 days after leision wasinduced, and unilateral rotational response was measured for 60 minutes(FIG. 16). This experiment uses a principle that, when the concentrationof dopamine in corpus striatum was decreased due to death of dopaminicneuronal cells, hypersensitivity of dopamine receptor was induced andapomorphine acts on dopamine receptor as agonist, thus excessivelyexciting hypersensitivity-induced corpus striatum. As a result, ananimal shows a rotational response in the direction opposite to impairedregion (Ungerstedt, Brain Res, 24, 485-493, 1970). Rotational responsewas measured by using automated rotometer referred to in theaforementioned Ungerstedt' journal, and net turns were calculated byusing the following formula:

net turns=contralateral turns−ipsilateral turns.

As shown in FIG. 16, normal control group, while administered with 0.2g/mL of ascorbinic acid in corpus striatum, did not show much change inunilateral net turns, leision-induced control group, administered onlywith dopamine hydroxide, showed significant increase (P<0.05).Meanwhile, target group, administered with silk peptide herein, showed asignificant decrease as compared to control group, and the effect wasverified to be enlarged as doses increased (P<0.05). Each value in FIG.16 refers to mean ±standard deviation.

Experimental Example III-3 Measurement of Amounts of Dopamine and ItsMetabolites in Corpus Striatum

To verify the effect of BG101 or BG201 on the concentration of dopamine,an important cause of Parkinson's disease, the concentrations ofdopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measuredin corpus striatum by using HPLC (Gilson, France) 2 weeks after BG101 orBG201 was administered and lesions were incurred, as set forth in theExperimental Example III-2.

A. Preparation of Tissue

Brain of a rat was extracted after putting its cerbical vertebral out ofjoint, and cut at intervals of 2 mm from optic chiasma by using a brainslicer (ZIVIC MILLER, US), and substantia nigra was separated withtissue punch. Anterior part of the remaining brain was divided into leftand right cerebral hemispheres on frozen glass plate. Only corpusstriatum was separated and extracted, and rapidly frozen with dry iceand kept at −70° C.

B. Analysis Method

After the frozen test subject was treated with 0.1 M of perchloric acidand 1 mM of EDTA, tissue homogenate was prepared with a sonicator andupper solution was obtained by centrifugal separation at 12,500 g for 20minutes. The upper solution was filtered onto nitrocellulose membranefilter paper (filter size 0.2 μm) and incorporated into HPLC. WATERSuBondapak™ C₁₈ 3.9×300 mm column (particle size 10 μm) was used, and amixture of 0.07 M sodium phosphate monobasic, 1 mM sodium octanesulfonicacid, 0.1 mM EDTA and 8% acetronitrile (pH 4.0) was used as a mobilephase at a rate of 0.7 mL/min. A predetermined dihydroxybenzylamine hadbeen added to measure the concentration of each material when preparingtissue homogenate as an internal standard material, and separatedmaterial was identified by using electrochemical analyzer.

C. Result of HPLC Analysis

As provided in FIG. 17, Dopamine ratio (ipsilateral/contralateral, %) incorpus striatum was significantly decreased in control group treatedwith only dopamine hydroxide as compared to sham control group (P<0.05).Meanwhile, in silk peptide treated group, the concentration of dopamineincreased significantly and in proportion to the dose of administration(P<0.05). Each value in FIG. 17 refers to mean ±standard deviation.

Parkinson's disease was incurred when dopamine was decreased in corpusstriatum due to destruction of dopamine neuronal cells in substantianigra. Decrease in concentration of dopamine induced by 6-OHDA is animportant sign of destruction of dopamine neuronal cell in substantianigra and dopamine neuronal fibril ends in corpus striatum. In fact,only about 10% of dopamine remains in corpus striatum in case of patientof last stage of Parkinson's disease as compared to healthy people.Therefore, silk peptide herein was physiologically and biochemicallyverified to inhibit the decrease of dopamine concentration, and thushave protective activity on dopamine neuronal cell.

Experimental Example III-4 Measurement of Lipid Peroxidation Level inCorpus Striatum (Malondialdehyde Experiment)

To verify how BG101 or BG201 inhibits lipid peroxidation, amount ofmalondialdehyde (MDA) was measured in corpus striatum 2 weeks afterBG101 or BG201 was administered and lesion was prepared as set forth inExperimental Example III-2.

A. Preparation of Tissue

Corpus striatum was extracted according to the aforementioned method,tissue homogenate was prepared by adding Krebs-Ringer Buffer (NaCl 120mM, KCl 4.8 mM, CaCl₂ 1.3 mM, MgSO₄ 1.2 mM, NaHCO₃ 25 mM, Glucose 6 mM,pH 7.6) and by using a sonicator.

B. Measurement of Lipid Peroxidation

As an index of lipid peroxiation, TBARS (Thiobarbituric acid reactivesubstances) concentration was measured according to Ohkawa's method(Anal Biochem. 95(2)351-8(1979)). After 8.1% SDS, 20% acetic acid (pH2.5), 0.8% Thiobarbituric acid (TBA) was added to a predetermined amountof tissue homogenate and BHT (2,6-di-t-butyl-p-cresol, 200 μM) was alsoadded to inhibit self-oxidation, reaction was performed at 100° C. for30 minutes. After the reactor was placed in ice water to rapidlyterminate the reaction, absorption was measured at 532 nm, and TEARSconcentration was calculated.

The concentration of MDA-TBA (Malonaldehyde-thiobarbituric acid) complexwas measured by using HPLC according to the method of Lazzarino et al.(Lazzarino et al., Free. Radic. Biol. Med. 13(5), 489-98, 1992) andChirico (1987). The specimen was injected into 5 μM Lichrosper 100 RP18column (4.6×250 mm), eluted by using 65% KH₂PO₄ (50 mM)/15% methanol/20%acetonitrile at a rate of 0.9 mL/min, and detected with UV/visibledetector (Hewlett-Packard, Series 1050) by using1,1,3,3-tetramethoxypropane (prepared in ethanol/water 40:60, v/v) as astandard specimen for indentifying peaks of BG101A-TBA complex.

C. Results

MDA generation ratio in corpus striatum (ipsilateral/contralateral, %)was shown as a ratio (%) of concentration of MDA-TBA complex to that ofTBARS in FIG. 18. Lipid peroxidation level in corpus striatumsignificantly increased in case of control group administered onlydopamine hydroxide as compared to sham control group (P<0.05).Meanwhile, silk peptide administered group showed significant decreasein lipid peroxidation level as compared to control group, and the degreeof decrease was proportion to the administration dose (P<0.05). Eachvalue in FIG. 18 refers to mean ±standard deviation.

As the most important cause of aging and degenerative disease, reactiveoxygen induces peroxidation of protein and lipid, weakens normalfunction of cells, and induces oxidative impairment and mutation of DNA.It was recently reported that anti-oxidative enzyme such as giutationperoxidase and catalase is decreased and hydroxyl radical ion isabnormally increase due to the increase of ferrous ion in patients ofParkinson's disease, and it indicates that oxidative stress in animportant factor for inducing Parkinson's disease (Ogawa, Eur. Neurol.34(suppl), 20-28, 1994). Therefore, it may be concluded that silkpeptide herein that can decrease lipid peroxidation degree has anactivity of suppressing aging process.

Experimental Example III-5 Measurement of Ratio of TH Immuno PositiveCells in Substantia Nigro (TH Immunohistochemical Experiment)

To verify the effect of BG101 or BG201 herein on parkinsonism animalmodel, TH immunohistochemical experiments were performed. 2 weeks afterBG101 or BG201 was administered and leision was prepared as set forth inExperimental Example III-2, TH immunohistochemical staining ofsubstantia nigro slice was performed and the number of stained THimmuno-positive cells was counted by using high-powered microscope.

A. Preparation of Tissue

As explained in Experimental Example II-2, after test animal wasadministered with BG101 or BG201 and lesion was induced, the test animalwas anaesthetized with 400 mg/mL of chloral hydrate, and 200 mL of 0.1 Mphosphoric acid buffer solution (PBS, pH 7.4) was reperfused throughheart, thus removing blood component in blood vessel, followed byreperfusion of 250-300 mL of fixing solution (4% paraformaldehyde/PBS).Brain was extracted and post-fixed with the fixing solution at 4° C. for15-24 hours. After washing the fixing solution with PBS, 10%, 20% and30% sucrose solutions were incorporated in this order, thus preventingice crystal. Brain tissue was embedded with embedding solution andquick-frozen in isopentane prefrozen with liquid nitrogen. Thecontinuous coronal slicing of brain tissue was performed into 10 μmthickness slices by using microtorning device (Cryostat; ReichertFrigocut model 2000), and placed in preserving solution comprising 30%glycerol, 30% ethylene glycol and 10% phosphate salt buffer solution(PB).

B. Immunohistochemical Analysis

Tissue was taken out of the preserving solution, washed with PBS 3 timesfor 10 minutes each time, and reacted with 5% hydrogen peroxide for 10minutes, followed by reaction with 10% normal goat serum (NGS), 3% BSA(Sigma) and 0.3% Triton X-100 for 30 minutes. It was reacted, in thisorder, with mouse αTH (dilution ratio=1:500, Boehringer Mannheim) as afirst antibody for one day, with biotinylated Vector laboratories (US)as a second antibody for 1 hour at room temperature, and withavidin-biotin-peroxidase composite (ABC, Vector laboratories, US),pre-diluted at a ratio of 1:100 before 30 minutes, at room temperaturefor 1 hour. The tissue was placed in DAB (diaminobenzidine 0.05%, H₂O₂0.003%) for 5 minutes for visualization, and was attached togelatin-coated slide and dried. It was dewatered by using 70%, 90% and100% ethanol, treated with xylene, sealed with covering glass andCanadian balsam, and observed, with microscope. In the aforementionedculture step, excess reagent was washed with PBS 3 times for 10 minuteseach time. Comparative staining was performed by using normal seruminstead of first and second antibodies.

C. Result of Immunohistochemical Analysis

The result of TH immununohistochemical staining with BG101 or BG201 incorpus striatum and substantia nigra and a variance of TH immunopositive cell ratio (ipsilateral/contralateral, %) in substantia nigrawere provided in FIGS. 19 and 20, respectively.

As shown in FIG. 19, in normal control group, TH immuno staining incorpus striatum and dopamine neuronal cell in substantia nigra wereclearly expressed. In contrast, in lesion control group, corpus striatumwas not stained in Doso-lateral area and most of dopamine neuronal cellsin substantia nigra par compacta were destroyed. Meanwhile, the groupadministered with silk peptide herein showed a significantly protectiveactivity on dopamine neuronal fibril ends and neuronal cells as comparedto control group.

As shown in FIG. 20, TH immuno positive cells were significantlydecreased in leision control group administered only with dopaminehydroxide as compared to normal control group (P<0.05). Meanwhile, thegroup administered with silk peptide herein was verified tosignificantly increase the TH immuno positive cells and the increase wasin proportion to administration dose (P<0.05). Each value in FIG. 20refers to mean ±standard deviation.

TH is an essential enzyme in generation of dopamine from tyrosine.TH-stained dopamine neuronal cells and ends may generate dopamine andform dopaminergic neural network. Thus, the aforementioned results showthat silk peptide herein has a protective activity for dopamine neuronalcells. Therefore, silk peptide herein may be used in functional food ordrug for preventing or treating degenerative cerebral disease,Parkinson's disease.

Experimental Example IV Effect of Concentration of Acetylcholine

Experiment was performed to verify that BG101 or BG201 herein inhibitsthe decrease of acetylcholine in brains of rats that was treated withamyloid β, thus serving a function of improving brain activity andinhibiting degenerative cerebral disease.

Experimental Example IV-1 Preparation of Animal Test Subject

Four Sprague Dawley rats (140-180 g, Dae Han BioLink Co., Korea) eachbox were placed under constant condition (temperature: 25±1° C.,relative humidity: 60±10%), and fed with unlimited water and food for 1week.

Experimental Example IV-2 Measurement of Concentration of Acetylcholine

The concentration of acetylcholine was measured by usingchemiluminescence according to methods of Islael and Lesbats (J.Neurochem. 37(6)1475-83(1981)). This method uses reactions thatacetylcholine was hydrolyzed into choline by esterase and furtherchanged to bestaine and hydrogen peroxide by choline oxidase. Hydrogenperoxide emits light when chemically reacted with luinol(5-amino-1,2,3,4-tetrahydro-1,4-phthalazinedione; Merck, Darmstadt,Germany) and peroxidase (Sigma, USA), and the amount of emitted lightmay be used to determine the concentration of acetylcholine.

While Aβ decreased about 75% of acetylcholine in Aβ treated controlgroup as compared to normal control group, the group treated with BG101or BG201 for 1 week significantly inhibited the decrease ofacetylcholine (P<0.05). Each value in FIG. 21 refers to mean ±standarddeviation.

Acetylcholine is neurotransmitter that is projected from basal gangliato cerebral cortex or hippocampus, thereby performing a very importantactivity for normal brain function (Richter et. al., Life Sci.19;26(20)1683-9(1980)). Especially, learning and memory has been knownto be varied by drug acting on acetylcholine system. Investigation ofpeople who died of Alzheimer type dementia has showed that theiracetykholinergic neuronal cells are much damaged. Choline agonists andcholine esterase inhibitors has been used for patients, as it isrecently known that the increase of acetylcholine acts in treating orpreventing dementia by improving cognitive function and inhibit thedevelopment of dementia. Up to present, there have been developedacetylcholine precursor such as Lecithin; receptor agonist such as RS-86and nicotine; and acetylcholine esterase inhibitor such as Tacrine andAricept, the former of which was approved by FDA and is on the Koreanmarket and the latter of which was also recently approved by FDA.However, their use is still open to argument because their effects donot last long and, weak and also seriously toxic.

The aforementioned results showed that silk peptide herein inhibits thedecrease of acetylcholine in brain, thus improving brain function andinhibiting degenerative cerebral disease.

Experimental Example V Depression Animal Model Experiment

Experimental Example V-1

Preparation of Animal Test Subject

Four Sprague Dawley rats (140-180 g, Dae Han BioLink Co., Korea) eachbox were placed under constant condition (temperature: 25±1° C.,relative humidity: 60±10%), and fed with unlimited water and food for 1week. Fifty rats were selected for experiment among the rats byexcluding ones that were undeveloped, show less motion or abnormalbehavior in forced swimming test.

Experimental Example V-2 Depression Animal Model Experiment

As a standard method for behavioral despair test, a forced swimming test(FST) was used in this experiment, which is known as principal test forverify anti-depression efficacy. FST test was performed as followsaccording to its proposer Porsolt et al. (Porsolt et al., Eur. J.Pharmacol. 51(3), 291-294, 1978).

A rat was placed for 15 minutes in a transparent cylindrical water tankwith 40 cm of height and 18 cm of diameter, which was filled with 15 cmdeep water. Although the rat violently tried to go out of the tank forthe first several minutes, the immobilization time increased as timewent by, and the rat maintained its body in the immobilization state forthe last several minutes. The immobilization state typically refers to astate that a rat pushes only its head above the surface and was in aminimum motion for keeping itself afloat. After forced swimming test,the rat was dried at 37° C. for 30 minutes and returned to the box.

After 24 hours, the second forced swimming was performed for 5 minutesunder the same condition with the first forced swimming test, and totalimmobilization time was measured. Due to learned helplessness, a ratusually shows more immobilization time compared to the first forcedswimming test. If a medicine decreases the immobilization time, it maybe considered to have an activity related to anti-depression function.Because some medicine loses its functions in a long-term administrationin spite of its efficacy in acute treatment and the anti-depressionactivity may be obtained after at least 2 weeks, the second forcedswimming test was performed after 7-day administration of medicine asusually done. The immobilization time was measured by recording totalprocess of the forced swimming and comparing between the control groupand the experiment group. Values measured by 3 different analyzers wereaveraged and used as an analysis data.

A. Administration Method

BG101 or BG201 herein was prepared into 100 mg/mL solution and orallyadministered. One time urgent administration was performed 1 hour beforethe second forced swimming, and long-term repeated administration wasperformed from 30 minutes after the first forced swimming for 7 days.

B. Anti-Depression Effect of One Time Urgent Administration

Anti-depression effect of one time urgent administration (1 g/kg) wasverified. Conventional drug, imipramine (Sigma, US), was used as apositive control group, and dose was determined as 20 mg/kg peradministration by referring to research results (Eur. J. Pharmacol.138(3), 413-416, 1987; Neuropharmacology 28(3), 229-233, 1989). Theresult was provided in FIG. 22.

Average immobilization time was significantly decreased by imipramineadministration compared to control group (*, P<0.05). Further, BG101 orBG201 administered group also showed a significant decrease inimmobilization time (**, P<0.05). Each value in FIG. 22 refers to mean±standard deviation.

C. Anti-Depression Effect of 7-Day Repeated Administration

Anti-depression effect of 7-day repeated administration (1 g/kg) wasverified. 50 mg/kg of BG101 or BG201 was orally administered one time aday for 7 days, and experiment was performed as aforementioned. Theresult was provided in FIG. 23.

Average immobilization time was significantly decreased by imipramineadministration compared to control group (P<0.05). Further, BG101 orBG201 administered group also showed a significant decrease inimmobilization time (P<0.05), Each value in FIG. 23 refers to mean±standard deviation.

From the results above, it may be verified that imipramine decreasedimmobilization time as compared to control group, and BG101 or BG201 hasalso an statistically significant anti-depression activity although itis lower than that of imipramine. However, considering side effect ofimipramine when administered long time, BG101 or BG201 has an advantagethat it may be administered in large amount for long time and thus mayhave superiority in preventing or treating depression.

Experimental Example VI Clinical Test for Improvement of Brain Function

Clinical test subjects were divided into two groups: (i) 67 of testgroup intaking capsule comprising 100 mg of BG-101 herein (“BF-7”) (33of BF-7 200 mg/day group & 34 of 400 mg/day group), (ii) 32 of placebogroup, and took capsules twice a day for 3 weeks.

Rey-Kim test was performed before and 3 weeks after intaking capsules,and the change was evaluated.

Rey-Kim test consists of Auditory Verbal Learning Test (AVLT) andComplex Figure test (CFT).

A. AVLT (1) Repeated Test

After words were mentioned each a second, test subject was required torecall the words, and test was repeated totally 5 times,

(2) Delayed Recall

After delayed period of 20 minutes, the test subject was required torecall the words again.

(3) Delayed Conformation

After delayed recall test, the subject test was given a paper andrequired to circle only the mentioned words.

B. CFT

(1) Drawing Test

A test subject was given CFT figures and a response paper before test,and required to draw the figures. After drawing, the CFT figures and thepaper were laid aside so that the test subject may not see them.

(2) Immediate Recall Test

The subject test was required to draw the figures immediately after thedrawing test.

(3) Delayed Recall Test

After delayed period of 20 minutes, the test subject was required todraw the figures again.

(4) Evaluation of CFT

Each of 18 items was evaluated considering its shape and place as fromzero to 2 points according to CFT evaluation standard. Thus, each testmay get from zero to 36 points.

C. Evaluation Item

{circle around (1)} Memory quotient (MQ): the most direct reflectiveindex of memorizing ability.

{circle around (2)} Memory maintenance: an index indicating how much andhow well memory is maintained precisely

{circle around (3)} Recall efficiency: an index indicating how preciselyand efficiently memory is utilized

{circle around (4)} Drawing/memory consensus: an index for determiningwhether an improved drawing result was caused by a drawing ability ormemory

{circle around (5)} Intelligence/memory consensus: an index indicating arelationship between enhancement of intelligence and enhancement ofmemory

D. Statistic Analysis of Data

The paired t-test was used to analyze difference between scores beforeand after administration. ANOVA was used to determine whether thedifferences of scores between the placebo, BF-7 200 mg, and BF-7 400 mggroups has statistical significance or not.

E. Result (1) Improvement of MQ by BF-7

Differences between MQ's before and after administration of the placebo,BF-7 200 mg and BF-7 400 mg groups are 3.1, 11.6 and 20.6, respectively.This result showed that the three cases all showed significantimprovement and that the improvement was in proportion to BF-7 dose(FIG. 24). MQ's were improved from 106 to 110, from 106.5 to 118, andfrom 106 to 126 in the cases of the placebo, BF-7 200 mg and BF-7 400 mggroups, respectively (FIG. 25).

(2) Improvement of Recall Efficiency by BF-7

A higher percentage score means better recall efficiency. While theplacebo group showed no significant improvement, BF-7 200 mg and BF-7400 mg groups showed significant improvement from 31% to 58.9% and from41.5% to 66.5%, respectively (FIG. 26).

(3) Improvement of Drawing/Memory Consensus by BF-7

As a percentage score is lower, a drawing result is more ascribed tolowered memory, While the placebo group showed no significantimprovement from 38% to 40% (P>0.05), BF-7 200 mg and BF-7 400 mg groupsshowed significant improvement from 36.8% to 56.5% and from 24.7% to65.2%, respectively, which showed that an improvement of intelligence isnot ascribed to drawing ability improvement but memory improvement (FIG.27).

(4) Improvement of Intelligence/Memory Consensus by BF-7

A higher percentage score means a better memory among people of same agewith similar intelligence. As shown in FIG. 28, the placebo and BF-7 200mg groups showed significant improvement from 55.5% to 63.4% and 52.9%to 78.9%, respectively. BF-7 400 group showed remarkable enhancementfrom 52.5% to 91.9% (P <0.05).

(5) Improvement of Memory Maintenance by BF-7

A higher percentage score means better memory maintenance. While theplacebo and BF-7 200 mg groups showed no significant improvement(P>0.05), BF-7 400 mg group showed significant improvement from 53.2% to61.3% (FIG. 29).

As set forth above, from the differences between MQ's before and afteradministration, it may be verified that all the three cases, theplacebo, BF-7 200 mg and BF-7 400 mg groups, showed significantimprovement of intelligence. Although the improvement in the placebogroup may be ascribed to wide range of standard deviation and, to someextent, a mental effect, as already reported, it can be concluded thatBF-7 has an activity of improving intelligence.

Further, from the fact that BF-7 also enhanced other index such asrecall efficiency and drawing/memory consensus, it may also concludethat BF-7 helps to improve recall ability, memory and learning abilityand effectively acts on neuronal degenerative disease such as dementia,thus being very invaluable material having a lot of scientific andindustrial uses.

As described above, the present invention provides a method ofefficiently preparing silk peptide having neuroprotective activity andlow molecular weight that can facilitates body's absorption.

Further, the present invention provides a composition comprising silkpeptide so produced for preventing or treating brain disease orimproving brain function. The composition of the present invention showsactivities or effects as set forth in Detailed Description and has muchlower side effect to human body because it comprises natural material,silk peptide, as an active ingredient.

1-52. (canceled)
 53. A method for improving a brain function, whichcomprises administering to a subject a food composition comprising asilk peptide having a weight average molecular weight of 200-100,000 asan active ingredient, wherein the brain function is learning and memoryability.
 54. The method according to claim 53, wherein the silk proteinis silk fibroin.
 55. The method according to claim 53, wherein the silkprotein has the weight average molecular weight of 200-15,000, and isproduced by hydrolysis using a calcium salt solution, acid hydrolysis,hydrolysis using a protease or a combination thereof, and.
 56. Themethod according to claim 55, wherein the protease is selected from thegroup consisting of trypsin, pepsin, alcalase, thermoase, flavourzyme,sumizyme, protamex, protin and a mixture thereof.
 57. The methodaccording to claim 56, wherein the protease is selected from the groupconsisting of thermoase, flavourzyme, sumizyme and a combinationthereof.
 58. The method according to claim 55, wherein the silk proteinhas the weight average molecular weight of 200-4,000 and is produced bysequentially performing (i) hydrolysis using the calcium salt solution,and (ii) hydrolysis using the protease selected from the groupconsisting of thermoase, flavourzyme, sumizyme and a mixture thereof.59. The method according to claim 58, wherein the silk protein has theweight average molecular weight of 200-2,000 and is produced bysequentially performing (i) hydrolysis using the calcium salt solution,and (ii) hydrolysis using a mixture of flavourzyme and sumizyme.
 60. Themethod according to claim 59, wherein the silk peptide has the weightaverage molecular weight of 200-1,200.
 61. The method according to claim59, wherein the calcium salt is calcium chloride.
 62. The methodaccording to claim 53, wherein the silk peptide is produced by acidhydrolysis and has the weight average molecular weight of 200-100,000.63. The method according to claim 62, wherein the silk peptide has theweight average molecular weight of 200-3,000.
 64. The method accordingto claim 63, wherein the silk peptide has the weight average molecularweight of 200-1,500.
 65. The method according to claim 53, wherein thesilk peptide has a neuroprotective activity.
 66. The method according toclaim 65, wherein the neuroprotective activity is accomplished byinhibiting a neuronal cell death.
 67. The method according to claim 66,wherein the inhibition of the neuron cell death is accomplished byinhibiting a neuronal apoptosis.
 68. A method for preventing or treatinga brain disease, which comprises administering to a subject apharmaceutical composition comprising a silk peptide having a weightaverage molecular weight of 200-100,000 as an active ingredient, whereinthe brain disease is Parkinson's disease, depression or ischemic stroke.69. The method according to claim 68, wherein the silk protein is silkfibroin.
 70. The method according to claim 68, wherein the silk proteinhas the weight average molecular weight of 200-15,000, and is producedby hydrolysis using a calcium salt solution, acid hydrolysis, hydrolysisusing a protease or a combination thereof, and.
 71. The method accordingto claim 70, wherein the protease is selected from the group consistingof trypsin, pepsin, alcalase, thermoase, flavourzyme, sumizyme,protamex, protin and a mixture thereof.
 72. The method according toclaim 71, wherein the protease is selected from the group consisting ofthermoase, flavourzyme, sumizyme and a combination thereof.
 73. Themethod according to claim 71, wherein the silk protein has the weightaverage molecular weight of 200-4,000 and is produced by sequentiallyperforming (i) hydrolysis using the calcium salt solution, and (ii)hydrolysis using the protease selected from the group consisting ofthermoase, flavourzyme, sumizyme and a mixture thereof.
 74. The methodaccording to claim 73, wherein the silk protein has the weight averagemolecular weight of 200-2,000 and is produced by sequentially performing(i) hydrolysis using the calcium salt solution, and (ii) hydrolysisusing a mixture of flavourzyme and sumizyme.
 75. The method according toclaim 74, wherein the silk peptide has the weight average molecularweight of 200-1,200.
 76. The method according to claim 74, wherein thecalcium salt is calcium chloride.
 77. The method according to claim 68,wherein the silk peptide is produced by acid hydrolysis and has theweight average molecular weight of 200-100,000.
 78. The method accordingto claim 77, wherein the silk peptide has the weight average molecularweight of 200-3,000.
 79. The method according to claim 78, wherein thesilk peptide has the weight average molecular weight of 200-1,500. 80.The method according to claim 68, wherein the silk peptide has aneuroprotective activity.
 81. The method according to claim 80, whereinthe neuroprotective activity is accomplished by inhibiting a neuronalcell death.
 82. The method according to claim 81, wherein the inhibitionof the neuron cell death is accomplished by inhibiting a neuronalapoptosis.